Systems and methods for fluorescence detection with a movable detection module

ABSTRACT

A fluorescence detection apparatus for analyzing samples located in a plurality of wells in a thermal cycler and methods of use are provided. In one embodiment, the apparatus includes a support structure attachable to the thermal cycler and a detection module movably mountable on the support structure. The detection module includes one or more channels, each having an excitation light generator and an emission light detector both disposed within the detection module. When the support structure is attached to the thermal cycler and the detection module is mounted on the support structure, the detection module is movable so as to be positioned in optical communication with different ones of the plurality of wells. The detection module is removable from the support structure to allow easy replacement.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/827,521,filed Jun. 30, 2010, entitled “Systems and Methods For FluorescenceDetection With A Movable Detection Module,” which is a continuation ofapplication Ser. No. 11/555,642, filed Nov. 1, 2006, entitled “Systemsand Methods For Fluorescence Detection With A Movable Detection Module,”which is a continuation of application Ser. No. 10/431,708, filed May 8,2003, entitled “Systems and Methods for Fluorescence Detection with aMovable Detection Module,” now U.S. Pat. No. 7,148,043. The respectivedisclosures of all applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates in general to fluorescence detectionsystems and in particular to a fluorescence detection system having amovable excitation/detection module for use with a thermal cycler.

Thermal cyclers are known in the art. Such devices are used in a varietyof processes for creation and detection of various molecules ofinterest, e.g., nucleic acid sequences, in research, medical, andindustrial fields. Processes that can be performed with conventionalthermal cyclers include but are not limited to amplification of nucleicacids using procedures such as the polymerase chain reaction (PCR). Suchamplification processes are used to increase the amount of a targetsequence present in a nucleic acid sample.

Numerous techniques for detecting the presence and/or concentration of atarget molecule in a sample processed by a thermal cycler are alsoknown. For instance, fluorescent labeling may be used. A fluorescentlabel (or fluorescent probe) is generally a substance which, whenstimulated by an appropriate electromagnetic signal or radiation,absorbs the radiation and emits a signal (usually radiation that isdistinguishable, e.g., by wavelength, from the stimulating radiation)that persists while the stimulating radiation is continued, i.e. itfluoresces. Some types of fluorescent probes are generally designed tobe active only in the presence of a target molecule (e.g., a specificnucleic acid sequence), so that a fluorescent response from a samplesignifies the presence of the target molecule. Other types offluorescent probes increase their fluorescence in proportion to thequantity of double-stranded DNA present in the reaction. These types ofprobes are typically used where the amplification reaction is designedto operate only on the target molecule.

Fluorometry involves exposing a sample containing the fluorescent labelor probe to stimulating (also called excitation) radiation, such as alight source of appropriate wavelength, thereby exciting the probe andcausing fluorescence. The emitted radiation is detected using anappropriate detector, such as a photodiode, photomultiplier,charge-coupled device (CCD), or the like.

Fluorometers for use with fluorescent-labeled samples are known in theart. One type of fluorometer is an optical reader, such as described byAndrews et al. in U.S. Pat. No. 6,043,880. A sample plate containing anarray of samples is inserted in the optical reader, which exposes thesamples to excitation light and detects the emitted radiation. Theusefulness of optical readers is limited by the need to remove thesample plate from the thermal cycler, making it difficult to monitor theprogress of amplification.

One improvement integrates the optical reader with a thermal cycler, sothat the sample plate may be analyzed without removing it from thethermal cycler or interrupting the PCR process. Examples of suchcombination devices are described in U.S. Pat. No. 5,928,907, U.S. Pat.No. 6,015,674, U.S. Pat. No. 6,043,880, U.S. Pat. No. 6,144,448, U.S.Pat. No. 6,337,435, and U.S. Pat. No. 6,369,863. Such combinationdevices are useful in various applications, as described, e.g., in U.S.Pat. No. 5,210,015, U.S. Pat. No. 5,994,056, U.S. Pat. No. 6,140,054,and U.S. Pat. No. 6,174,670.

Existing fluorometers suffer from various drawbacks. For instance, insome existing designs, different light sources and detectors areprovided for different sample wells in the array. Variations among thelight sources and/or detectors lead to variations in the detectedfluorescent response from one well to the next. Alternatively, the lightsource and/or detector may be arranged in optical communication withmore than one of the wells, with different optical paths to and/or fromeach well. Due to the different optical paths, the detected fluorescentresponse varies from one sample well to the next. To compensate for suchvariations, the response for each sample well must be individuallycalibrated. As the number of sample wells in an array increases, thisbecomes an increasingly time-consuming task, and errors in calibrationmay introduce significant errors in subsequent measurements.

In addition, existing fluorometers generally are designed such that thelight sources and detectors are fixed parts of the instrument. Thislimits an experimenter's ability to adapt a fluorometer to a differentapplication. For instance, detecting a different fluorescent labelgenerally requires using a different light source and/or detector. Manyexisting fluorometers make it difficult for an experimenter toreconfigure light sources or detectors, thus limiting the variety offluorescent labels that may be used.

It is also difficult to perform concurrent measurements of a number ofdifferent fluorescent labels that may be present in a sample (or indifferent samples). As described above, to maximize the data obtained inan assay, experimenters often include multiple fluorescent labelingagents that have different excitation and/or emission wavelengths. Eachlabeling agent is adapted to bind to a different target sequence, inprinciple allowing multiple target sequences to be detected in the samesample. Existing fluorometers, however, do not facilitate suchmultiple-label experiments. Many fluorometers are designed for a singlecombination of excitation and emission wavelengths. Others providemultiple light sources and detectors to allow detection of multiplelabels; however, these configurations often allow only one label to beprobed at a time because the excitation wavelength of one label mayoverlap the emission wavelength of another label; excitation lightentering the detector would lead to incorrect results. Probing multiplelabels generally cannot be done in parallel, slowing the data collectionprocess.

Therefore, an improved fluorometer for a thermal cycler that overcomesthese disadvantages would be desirable.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention provide fluorescence detection in athermal cycling apparatus. According to one aspect of the invention, afluorescence detection apparatus for analyzing samples located in aplurality of wells in a thermal cycler includes a support structureattachable to the thermal cycler and a detection module movablymountable on the support structure. The detection module includes anexcitation light generator and an emission light detector, both disposedwithin the detection module. When the support structure is attached tothe thermal cycler and the detection module is mounted on the supportstructure, the detection module is movable so as to be positioned inoptical communication with different ones of the plurality of wells.

According to another aspect of the invention, the detection module mayinclude two or more excitation light generators and two or more emissionlight detectors arranged to form two or more excitation/detection pairs.In one embodiment, the excitation/detection pairs are arranged such thateach excitation/detection pair is simultaneously positionable in opticalcontact with a different one of the plurality of wells. In analternative embodiment, excitation/detection pairs are arranged suchthat when a first one of the excitation/detection pairs is positioned inoptical contact with any one of the plurality of wells, a different oneof the excitation/detection pairs is not in optical contact with any oneof the plurality of wells. In some embodiments, the detection module isdetachably mounted on the support structure, thereby enabling a user toreplace the detection module with a different detection module.

According to yet another aspect of the invention, a method for detectingthe presence of a target molecule in a solution is provided. A pluralityof samples is prepared, each sample containing a fluorescent probeadapted to bind to a target molecule. Each sample is placed in arespective one of a number of sample wells of a thermal cyclerinstrument, the thermal cycler instrument having a detection modulemovably mounted therein, the detection module including anexcitation/detection channel, the excitation/detection channel includingan excitation light generator disposed within the detection module andan emission light detector disposed within the detection module. Thethermal cycler instrument is used to stimulate a reaction, and thesample wells are scanned to detect a fluorescent response by moving thedetection module and activating the excitation/detection channel. Duringthe scanning, the detection module is moved such that theexcitation/detection channel is sequentially positioned in opticalcommunication with each of the plurality of sample wells. Where thedetection module includes multiple excitation/detection pairs orchannels, channels may be active in parallel or sequentially.

The following detailed description together with the accompanyingdrawings will provide a better understanding of the nature andadvantages of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a thermal cycling apparatus according toan embodiment of the present invention;

FIG. 2 is an exploded view of a lid assembly for a thermal cyclingapparatus according to an embodiment of the present invention;

FIG. 3 is a bottom view of a fluorometer assembly for a thermal cyclingapparatus according to an embodiment of the present invention;

FIG. 4 is a top view of detection module according to an embodiment ofthe present invention;

FIGS. 5A-B are bottom views of detection modules according toalternative embodiments of the present invention;

FIG. 6 is a schematic diagram of an excitation/detection pair for adetection module according to an embodiment of the present invention;

FIG. 7 is a block diagram illustrating electrical connections for a lidassembly for a thermal cycling apparatus according to an embodiment ofthe present invention; and

FIG. 8 is a flow diagram of a process for using a thermal cycler havinga fluorescence detection system according to an embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

An exemplary apparatus embodiment of the present invention will bedescribed with reference to the accompanying drawings, in which likereference numerals indicate corresponding parts. Methods of using theapparatus will also be described. It is to be understood thatembodiments shown and described herein are illustrative and not limitingof the invention.

I. Exemplary Apparatus

FIG. 1 is a perspective view of a thermal cycling apparatus 100according to an embodiment of the present invention. Apparatus 100consists of a base unit 110 and a lid assembly 112. Base unit 110, whichmay be of conventional design, provides power and control functions fora thermal cycling process via conventional electronic components (notshown), such as programmable processors, clocks, and the like. Base unit110 also provides a user interface 116 that may include a keypad 118 andan LCD display screen 120, enabling a user to control and monitoroperation of the thermal cycler. Base unit 110 connects to an externalpower source (e.g., standard 120 V ac power) via a power cable 121. Someexamples of base unit 110 include the DNA Engine®, Dyad™, and Tetrad™thermal cyclers sold by MJ Research, Inc., assignee of the presentapplication.

Lid assembly 112 includes a sample unit and a fluorescence detectionapparatus, disposed within a lid 122; these components will be describedbelow. Lid 122 has a handle 124 to aid in its placement on and removalfrom base unit 110, and ventilation holes 126. Lid 122 provides opticaland thermal isolation for the components inside lid assembly 112.

FIG. 2 is an exploded view of the inside of lid assembly 112. Shown area sample unit 202, a lid heater 204, and a fluorometer assembly 206.Sample unit 202 contains a number of sample wells 210 arranged in aregular array (e.g., an 8×12 grid). In one embodiment, each sample well210 holds a removable reaction vessel (not shown), such as a tube, thatcontains a nucleic acid sample to be tested, together with appropriatePCR reactants (buffers, primers and probes, nucleotides, and the like)including at least one fluorescent label or probe adapted to bind to orotherwise respond to the presence of a target nucleic acid sequence. Thereaction vessels are advantageously provided with transparent samplecaps (not shown) that fit securely over the tops of the vessels toprevent cross-contamination of samples or spillage during handling.Reaction vessels may also be sealed in other ways, including the use offilms such as Microseal®B (made by MJ Research, Inc.), wax products suchas Chill-out™ (made by MJ Research, Inc.), or mineral oil. In analternative configuration, a removable sample tray (not shown) thatholds one or more distinct samples at locations corresponding to samplewells 210 is used. The sample tray may also be sealed in any of the waysdescribed above.

Sample unit 202 also includes heating elements (e.g., Peltier-effectthermoelectric devices), heat exchange elements, electrical connectionelements for connecting the heating elements to base unit 110, andmechanical connection elements. These components (not shown) may be ofconventional design. Sample unit 202 also provides electricalconnections for lid heater 204 and fluorometer assembly 206 viamultiwire cables 212, which are detachably connected to connectors 214.

Lid heater 204 has holes 220 therethrough, matching the size and spacingof the sample wells 210, and electronically controlled heating elements(not shown). Lid heater 204 is coupled to lid 122. The couplingmechanism (not shown) is advantageously movable (e.g., lid heater 204may be attached to lid 122 by a hinge) in order to provide access tofluorometer assembly 206 when lid 122 is removed from sample unit 202.When lid 122 is in place on sample unit 202, supports 224 hold lidheater 204 in position. Lower portions 226 of supports 224 areadvantageously designed to compress lid heater 204 toward sample unit202, thereby reducing the possibility of sample evaporation duringoperation of apparatus 100. This compression also allows reactionvessels of different sizes to be used. Lid heater 204 is used to controlthe temperature of the sample caps (or other sealants) of reactionvessels sample wells 210, in order to prevent condensation from formingon the caps during thermal cycling operation.

Lid heater 204 advantageously includes one or more calibration elements222 positioned between selected ones of holes 220 or in other locationsaway from the holes, such as near the periphery of lid heater 204.Calibration elements 222 provide a known fluorescence response and maybe used to calibrate fluorescence detectors in fluorometer assembly 206.Calibration elements 222 may be made, e.g., of a fluorescent coating ona glass or plastic substrate, or they may consist of a plastic with adye impregnated in it, fluorescent glass, or a fluorescent plastic suchas polyetherimide (PEI). Neutral-density or other types of filters maybe placed over the fluorescent material in order to avoid saturating thefluorescence detectors. In general, any material may be used, providedthat its fluorescence characteristics are sufficiently stable over timewith the application of light (photo-bleaching) and heat. To the extentpractical, the effect of temperature on the fluorescence response isadvantageously minimized. Where multiple calibration elements 222 areprovided, different materials may be used for different ones of thecalibration elements. In an alternative embodiment, lid heater 204 maybe omitted, and calibration elements 222 may be disposed on the surfaceof sample unit 202.

Sample unit 202 and lid heater 204 may be of conventional design.Examples of suitable designs include sample unit and lid heatercomponents of the various Alpha™ modules sold by MJ Research, Inc.,assignee of the present application.

Fluorometer assembly 206 includes a support frame or platform 230fixedly mounted inside lid 122. Movably mounted on the underside ofsupport frame 230 is a shuttle 232, which holds a detection module 234.Shuttle 232 is movable in two dimensions so as to position detectionmodule 234 in optical communication with different ones of the samplewells 210 in sample unit 202 through the corresponding holes 220 in lidheater 204. Support frame 230 and supports 224 are advantageouslydimensioned such that when lid 122 is positioned in base unit 110 andclosed, detection module 234 is held in close proximity to lid heater204; one of skill in the art will appreciate that this arrangementreduces light loss between the sample wells and the detection module.

FIG. 3 is a bottom view of fluorometer assembly 206, showing a movablemounting of shuttle 232 and detection module 234. In this embodiment,translation stages driven by stepper motors are used to move the shuttle232, to which detection module 234 is detachably coupled, to a desiredposition. Specifically, support platform 230 has an x-axis stepper motor302 and a lead screw 304 attached thereto. Stepper motor 302 operates toturn lead screw 304, thereby moving a translation stage 306 along the xdirection (indicated by arrow). Limit switches 308 are advantageouslyprovided to restrict the motion of translation stage 306 to anappropriate range, large enough to allow detection module 234 to beplaced in optical contact with any of the wells while preventingtranslation stage 306 from contacting other system components, such asstepper motor 302.

Translation stage 306 has a y-axis stepper motor 316 and a lead screw318 mounted thereon. Stepper motor 316 operates to turn lead screw 318,thereby moving shuttle 232 along the y direction (indicated by arrow).Limit switches 320 are advantageously provided to restrict the motion ofshuttle 232 to an appropriate range, large enough to allow detectionmodule 234 to be placed in optical contact with any of the wells, whilepreventing shuttle 232 from contacting other system components, such asstepper motor 316.

Stepper motors 302, 316, lead screws 304, 318, and limit switches 308,320 may be of generally conventional design. It will be appreciated thatother movable mountings may be substituted. For example, instead ofdirectly coupling the motors to the lead screws, indirect couplings suchas chain drives or belt drives may be used. Chain drives, belt drives,or other drive mechanisms may also be used to position the detectionmodule without lead screws, e.g., by attaching a translation stage tothe chain, belt, or other drive mechanism. Other types of motors, suchas servo motors or linear motors, may also be used. Different drivemechanisms may be used for different degrees of freedom.

Shuttle 232 holds detection module 234 via connectors 330, 331.Connectors 330, 331 which may vary in design, are configured to supportand align detection module 234 on the underside of shuttle 232. Theconnectors are advantageously adapted to allow easy insertion andremoval of detection module 234, to facilitate replacement of thedetection module. In one embodiment, connectors 330 provide mounting fora cylindrical member (not shown) that pivotably holds an edge ofdetection module 234, while connectors 331 include ball plungers mountedon shuttle 232 that are insertable into corresponding receptacles ondetection module 234. Electrical connections (not shown) between shuttle232 and detection module 234 may also be provided, as will be describedbelow.

FIG. 4 is a top view of detection module 234. Detection module 234includes fittings 420 that couple to corresponding connectors 330 on theunderside of shuttle 232, thereby securing detection module 234 in placeso that it moves as a unit with shuttle 232. Detection module 234 alsoincludes an electrical connector 424 that couples to a correspondingelectrical connector on the underside of shuttle 232, thereby allowingcontrol and readout signals to be provided to and obtained fromdetection module 234.

FIG. 5A is a bottom view of one embodiment of detection module 234,showing four openings 502, 504, 506, 508 for four independentlycontrolled fluorescent excitation/detection channels (also referred toas “excitation/detection pairs”) arranged inside the body of detectionmodule 234. Examples of excitation/detection channels will be describedbelow. The spacing of openings 502, 504, 506, 508 corresponds to thespacing of sample wells 210. Thus, when opening 502 is placed in opticalcommunication with one of the sample wells 210, openings 504, 506, and508 are each in optical communication with a different one of the samplewells 210. Openings 502, 504, 506, 508 may simply be holes through thebottom surface of detection module 234, or they may be made of anysubstance that has a high degree of transparency to the excitation anddetection light wavelengths of their respective channels.

FIG. 5B is a bottom view of a detection module 234′ according to analternative embodiment of the invention. In this embodiment, fouropenings 512, 514, 516, 518 are provided, but they are arranged in astaggered fashion so that only one opening at a time may be in opticalcommunication with any of the sample wells. This configuration is usefulfor reducing cross-talk between the excitation/detection pairs.

FIG. 6 is a schematic diagram illustrating a configuration of opticalelements for an excitation/detection channel (or excitation/detectionpair) 600 according to an embodiment of the invention. Detection module234 may include one or more instances of excitation/detection pair 600,each of which provides an independent fluorescence detection channel.Excitation/detection pair 600 is arranged inside opaque walls 602, whichprovide optical isolation from other excitation/detection pairs that maybe included in detection module 234, as well as from external lightsources. An excitation light path 604 includes a light-emitting diode(LED) or other light source 606, a filter 608, a lens 610, and a beamsplitter 612. A detection light path 620 includes beam splitter 612, afilter 624, a lens 626, and a photodiode or other photodetector 628.Beam splitter 612 is advantageously selected to be highly transparent tolight of the excitation wavelength and highly reflective of light at thedetection (fluorescent response) wavelength.

The components of excitation light path 604 are arranged to directexcitation light of a desired wavelength into a reaction vessel 616 heldin a sample well 210 of sample block 202. The desired wavelength dependson the particular fluorescent labeling agents included in reactionvessel 616 and is controlled by selection of an appropriate LED 606 andfilter 608. Optical communication between the excitation/detection pair600 and reaction vessel 616 is provided by opening 502 in opaque walls602 and a hole 220 through lid heater 204, as described above. Tomaximize light transmission to and from excitation/detection pair 600,the space between opening 502 and lid heater 204 is advantageously madesmall during operation.

Excitation light that enters reaction vessel 616 excites the fluorescentlabel or probe therein, which fluoresces, thereby generating light of adifferent wavelength. Some of this light exits reaction vessel 616 ondetection light path 620 and passes through opening 502. Beam splitter612 directs a substantial portion of the fluorescent light throughfilter 624, which filters out the excitation frequency, and lens 626,which focuses the light onto the active surface of photodiode 628.Photodiode 628 generates an electrical signal in response to theincident light. This electrical signal is transmitted by a readoutsignal path 630 to circuit board 634, which routes the signal toelectrical connector 424 for readout. Circuit board 634 and/or signalpath 630 may also include other components, such as pre-amplifiers, forshaping and refining the electrical signal from photodiode 628.

LED 606 and photodiode 628 may be controlled by signals received viaconnector 424, as indicated by respective control signal paths 636, 638.Control signals for LED 606 may operate to activate and deactivate LED606 at desired times; control signals for photodiode 628 may operate toactivate and deactivate photodiode 628 at desired times, adjust a gainparameter, and so on.

While FIG. 6 shows one excitation/detection pair 600, it is to beunderstood that an embodiment of detection module 234 may contain anynumber of such pairs, each of which is advantageously in opticalisolation from the others and has its own opening for opticalcommunication with the sample wells (e.g., openings 504, 506, 508 ofFIG. 5). The various excitation/detection pairs are independentlycontrolled and independently read out, but their respective control andreadout paths may all be coupled to circuit board 634.

The configuration of excitation/detection pairs may be varied from thatshown, and the excitation and detection light paths may includeadditional components, fewer components, or any combination of desiredcomponents. The optics may be modified as appropriate for a particularapplication (e.g., the optical path may be shorter in embodiments wherelid heater 204 is not included) and use any number and combination ofcomponents including but not limited to lenses, beam splitters, mirrors,and filters. While LEDs provide a compact and reliable light source, useof other types of coherent or incoherent light sources, such as laserdiodes, flash lamps, and so on, is not precluded. Similarly, thedetectors are not limited to photodiodes; any type of photodetector maybe substituted, including photomultipliers and charge-coupled devices(CCDs). Each excitation/detection pair is advantageously configured as aself-contained assembly, requiring only external electrical connectionsto make it operational. Because the length of the excitation anddetection optical paths do not vary from one experiment to the next, itis desirable to fixedly mount and optimize the various opticalcomponents of each excitation/detection pair 600 inside detection module234 during manufacture so that further adjustments during operation arenot required.

FIG. 7 is a block diagram illustrating electrical connections for lidassembly 112. A main processing board 702 is mounted in lid assembly112. Main processing board 702 includes a primary signal processor 704,a stepper motor driver unit 706, a connection 708 for electrical power,and a connection 710 for an external computer (e.g., a personalcomputer, or PC). Main processing board 702 also provides connectors 214for cables 212 that provide transmission of electrical signals to andfrom lid 122.

Lid 122 includes a secondary processing board 720 that facilitatescommunication between main processing board 702 and stepper motors 302,316, as well as shuttle 232. Secondary processing board 720 includesconnectors 722 for cables 212, a connector 724 that connects a cable 726to shuttle 232, and connectors 732 and 734 for cables 736, 738 thatprovide control signals to the x and y stepper motors 302, 316. Routingpaths (not shown) in secondary processing board 720 establishappropriate signal connections between the various connectors.

Cable 726 is used to communicate control signals for detection module234, such as activating and deactivating individual light sources, andto receive signals from the photodetectors included in detection module234. Electrical connector 730 is provided on shuttle 232 for passingsignals to and from detection module 234. Electrical connector 730accepts the mating connector 424 on the top surface of detection module234 when detection module 234 is mounted on shuttle 232. In analternative embodiment, cable 726 may attach directly to detectionmodule 234.

As mentioned above, main processing board 702 provides a connection 710to an external computer (not shown). The external computer may be usedto control the motion of shuttle 232 and the operation of detectionmodule 234, as well as for readout and analysis of fluorometry dataobtained from detection module 234.

As described above, detection module 234 is designed to beself-contained and detachable from shuttle 232. This allows for areconfigurable fluorometry system, in which an experimenter is able tochange detection modules as desired to perform different measurements.For instance, different detection modules may be optimized for differentfluorescent labeling agents (or combinations of agents). If theexperimenter wishes to study a different agent, she simply installs theappropriate detection module. Installation is a matter of attachingelectrical connector 424 and mechanical connectors 420 on the top of thedesired detection module 234 to corresponding connectors on theunderside of shuttle 234. In some embodiments, the connectors aredesigned such that the electrical connection is made automatically asthe mechanical connection is engaged. As noted above, lid heater 204 isadvantageously movably mounted so as to allow access to fluorescenceassembly 230, thereby allowing experimenters to change detectionmodules.

It will be appreciated that the apparatus described herein isillustrative and that variations and modifications are possible. Forinstance, the base and sample unit may be designed as an integratedsystem or separated further into smaller modular components. Thefluorometer assembly need not be attached or otherwise integrated intothe lid, so long as it is mountable in a fixed position relative to thesample wells. Any mechanism may be used to make the detection modulemovable so as to position it in optical communication with differentones of the sample wells, not limited to translation stages or steppermotors. The detection module may include any number (one or more) ofexcitation/detection pairs operable as independent detection channels,and different pairs may be designed to detect the same fluorescent probeor different fluorescent probes. In one alternative embodiment, thedetection module includes a row of excitation/detection pairs withoptical windows arranged to correspond to a row of the sample array, andthe detection module is made movable in one direction to interrogatedifferent columns of the array.

The external computer is also optional, and any of its functions may beintegrated into the thermal cycler device; conversely, control functionsfor the thermal cycler may be implemented to operate on the externalcomputer, thereby providing a single control device for the entireapparatus. In one embodiment, the external computer is used to controlthe position of detection module 234 with respect to the sample wellsand operations of the light source(s) and detector(s). In addition, anyexternal computer may be special purpose control and signal-processingdevices as well as a general-purpose computer such as a PC.

II. Methods of Use

The apparatus described herein can be used to detect the amount ofamplification product generated in an amplification reaction bydetecting the amount of fluorescence. Various amplification techniquescan be used to quantify target sequences present in DNA or RNA samples.Such techniques, which involve enzymatic synthesis of nucleic acidamplicons (copies) that contain a sequence that is complementary to thesequence being amplified, are well known in the art and widely used.These include, but are not limited to the polymerase chain reaction(PCR), RT-PCR, strand displacement amplification (SDA), transcriptionbased amplification reactions, ligase chain reaction (LCR), and others(see, e.g. Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual,1995; U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications, Innis et al., eds, 1990; Walker, et al.,Nucleic Acids Res. 20(7):1691-6, 1992; Walker, PCR Methods Appl3(1):1-6, 1993; Phyffer, et al., J. Clin. Microbiol. 34:834-841, 1996;Vuorinen, et al., J. Clin. Microbiol. 33:1856-1859, 1995; Compton,Nature 350(6313):91-2, 1991; Lisby, Mol. Biotechnol. 12(1):75-991999;Hatch et al., Genet. Anal. 15(2):35-40, 1999; and Iqbal et al., Mol.Cell. Probes 13(4):315-320; 1999). Nucleic acid amplification isespecially beneficial when the amount of target sequence present in asample is very low. By amplifying the target sequence and detecting theamplicon synthesized, the sensitivity of an assay may be vastlyimproved, since fewer copies of the target sequence are needed at thebeginning of the assay to better ensure detection of nucleic acid in thesample belonging to the organism or virus of interest.

Measurement of amplification products can be performed after thereaction has been completed or in real time (i.e., substantiallycontinuously). If measurement of accumulated amplified product isperformed after amplification is complete, then detection reagents(e.g., fluorescent probes) can be added after the amplificationreaction. Alternatively, probes can be added to the reaction prior to orduring the amplification reaction, thus allowing for measurement of theamplified products either after completion of amplification or in realtime. If amplified products are measured in real time, initial copynumber can be estimated by determining the cycle number at which thesignal crosses a threshold and projecting back to initial copy number,assuming exponential amplification.

A. Fluorescent Probes

A number of formats are available that make use of fluorescent probes.These formats are often based on fluorescence resonance energy transfer(FRET) and include molecular beacon, and TaqMan® probes. FRET is adistance-dependent interaction between a donor and acceptor molecule.The donor and acceptor molecules are fluorophores. If the fluorophoreshave excitation and emission spectra that overlap, then in closeproximity (typically around 10-100 angstroms) the excitation of thedonor fluorophore is transferred to the acceptor fluorophore. As aresult, the lifetime of the donor molecule is decreased and itsfluorescence is quenched, while the fluorescence intensity of theacceptor molecule is enhanced and depolarized. When the excited-stateenergy of the donor is transferred to a non-fluorophore acceptor, thefluorescence of the donor is quenched without subsequent emission offluorescence by the acceptor. In this case, the acceptor functions as aquenching reagent.

One FRET-based format for real-time PCR uses DNA probes known as“molecular beacons” (see, e.g., Tyagi et al., Nat. Biotech. 16:49-53,1998; U.S. Pat. No. 5,925,517). Molecular beacons have a hairpinstructure wherein the quencher dye and reporter dye are in intimatecontact with each other at the end of the stem of the hairpin. Uponhybridization with a complementary sequence, the loop of the hairpinstructure becomes double stranded and forces the quencher and reporterdye apart, thus generating a fluorescent signal. A related detectionmethod uses hairpin primers as the fluorogenic probe (Nazarenko et al.,Nucl. Acid Res. 25:2516-2521, 1997; U.S. Pat. No. 5,866,336; U.S. Pat.No. 5,958,700). The PCR primers can be designed in such a manner thatonly when the primer adopts a linear structure, i.e., is incorporatedinto a PCR product, is a fluorescent signal generated.

Amplification products can also be detected in solution using afluorogenic 5′ nuclease assay, a TaqMan assay. See Holland et al., Proc.Natl. Acad. Sci. U.S.A. 88: 7276-7280, 1991; U.S. Pat. Nos. 5,538,848,5,723,591, and 5,876,930. The TaqMan probe is designed to hybridize to asequence within the desired PCR product. The 5′ end of the TaqMan probecontains a fluorescent reporter dye. The 3′ end of the probe is blockedto prevent probe extension and contains a dye that will quench thefluorescence of the 5′ fluorophore. During subsequent amplification, the5′ fluorescent label is cleaved off if a polymerase with 5′ exonucleaseactivity is present in the reaction. The excising of the 5′ fluorophoreresults in an increase in fluorescence which can be detected.

In addition to the hairpin and 5′-nuclease PCR assay, other formats havebeen developed that use the FRET mechanism. For example, single-strandedsignal primers have been modified by linkage to two dyes to form adonor/acceptor dye pair in such a way that fluorescence of the first dyeis quenched by the second dye. This signal primer contains a restrictionsite (U.S. Pat. No. 5,846,726) that allows the appropriate restrictionenzyme to nick the primer when hybridized to a target. This cleavageseparates the two dyes and a change in fluorescence is observed due to adecrease in quenching. Non-nucleotide linking reagents to coupleoligonucleotides to ligands have also been described (U.S. Pat. No.5,696,251).

Other amplification reactions that can be monitored using a fluorescentreading include those that are quantified by measuring the amount ofDNA-binding dye bound to the amplification product. Such assays usefluorescent dyes, e.g., ethidium bromide or SYBR Green I (MolecularProbes, Inc., Eugene, Oreg.; U.S. Pat. Nos. 5,436,134 and 5,658,751)that exhibit increased fluorescence when intercalated into DNA (see,e.g., U.S. Pat. Nos. 5,994,056 and 6,171,785). Use of SYBR Green I forthis purpose is also described in Morrison et al. (Biotechniques 24,954-962, 1998). An increase in fluorescence reflects an increase in theamount of double-stranded DNA generated by the amplification reaction.

Other fluorescent probes include inorganic molecules, multi-molecularmixtures of organic and/or inorganic molecules, crystals,heteropolymers, and the like. For example, CdSe-CdS core-shellnanocrystals enclosed in a silica shell may be easily derivatized forcoupling to a biological molecule (Bruchez et al. (1998) Science, 281:2013-2016). Similarly, highly fluorescent quantum dots (zincsulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

Multiplex assays may also be performed using apparatus 100. MultiplexPCR results in the amplification of multiple polynucleotide fragments inthe same reaction. See, e.g., PCR PRIMER, A LABORATORY MANUAL(Dieffenbach, ed. 1995) Cold Spring Harbor Press, pages 157-171. Forinstance, different target templates can be added and amplified inparallel in the same reaction vessel. Multiplex assays typically involvethe use of different fluorescent labels to detect the different targetsequences that are amplified.

B. PCR Conditions and Components

Exemplary PCR reaction conditions typically comprise either two or threestep cycles. Two step cycles have a denaturation step followed by ahybridization/elongation step. Three step cycles comprise a denaturationstep followed by a hybridization step followed by a separate elongationstep. The polymerase reactions are incubated under conditions in whichthe primers hybridize to the target sequences and are extended by apolymerase. The amplification reaction cycle conditions are selected sothat the primers hybridize specifically to the target sequence and areextended.

Successful PCR amplification requires high yield, high selectivity, anda controlled reaction rate at each step. Yield, selectivity, andreaction rate generally depend on the temperature, and optimaltemperatures depend on the composition and length of the polynucleotide,enzymes and other components in the reaction system. In addition,different temperatures may be optimal for different steps. Optimalreaction conditions may vary, depending on the target sequence and thecomposition of the primer. Thermal cyclers such as apparatus 100 providethe necessary control of reaction conditions to optimize the PCR processfor a particular assay. For instance, apparatus 100 may be programmed byselecting temperatures to be maintained, time durations for each cycle,number of cycles, and the like. In some embodiments, temperaturegradients may be programmed so that different sample wells may bemaintained at different temperatures, and so on.

Fluorescent oligonucleotides (primers or probes) containing base-linkedor terminally-linked fluors and quenchers are well-known in the art.They can be obtained, for example, from Life Technologies (Gaithersburg,Md.), Sigma-Genosys (The Woodlands, Tex.), Genset Corp. (La Jolla,Calif.), or Synthetic Genetics (San Diego, Calif.). Base-linked fluorsare incorporated into the oligonucleotides by post-synthesismodification of oligonucleotides that are synthesized with reactivegroups linked to bases. One of skill in the art will recognize that alarge number of different fluorophores are available, including fromcommercial sources such as Molecular Probes, Eugene, Oreg. and otherfluorophores are known to those of skill in the art. Useful fluorophoresinclude: fluorescein, fluorescein isothiocyanate (FITC), carboxytetrachloro fluorescein (TET), NHS-fluorescein, 5 and/or 6-carboxyfluorescein (FAM), 5-(or 6-) iodoacetamidofluorescein, 5-{[2 (and3)-5-(Acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein),and other fluorescein derivatives, rhodamine, Lissamine rhodamine Bsulfonyl chloride, Texas red sulfonyl chloride, 5 and/or 6 carboxyrhodamine (ROX) and other rhodamine derivatives, coumarin,7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA),and other coumarin derivatives, BODIPY™ fluorophores, Cascade Blue™fluorophores such as 8-methoxypyrene-1,3,6-trisulfonic acid trisodiumsalt, Lucifer yellow fluorophores such as3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins derivatives,Alexa fluor dyes (available from Molecular Probes, Eugene, Oreg.) andother fluorophores known to those of skill in the art. For a generallisting of useful fluorophores, see also Hermanson, G. T., BIOCONJUGATETECHNIQUES (Academic Press, San Diego, 1996).

The primers for the amplification reactions are designed according toknown algorithms. For example, algorithms implemented in commerciallyavailable or custom software can be used to design primers foramplifying the target sequences. Typically, the primers are at least 12bases, more often 15, 18, or 20 bases in length. Primers are typicallydesigned so that all primers participating in a particular reaction havemelting temperatures that are within 5° C., and most preferably within2° C. of each other. Primers are further designed to avoid priming onthemselves or each other. Primer concentration should be sufficient tobind to the amount of target sequences that are amplified so as toprovide an accurate assessment of the quantity of amplified sequence.Those of skill in the art will recognize that the amount ofconcentration of primer will vary according to the binding affinity ofthe primers as well as the quantity of sequence to be bound. Typicalprimer concentrations will range from 0.01 μM to 0.5 μM.

One of skill in the art will further recognize that it is desirable todesign buffer conditions to allow for the function of all reactions ofinterest. Thus, buffer conditions can be designed to support theamplification reaction as well as any enzymatic reactions associatedwith producing signals from probes. A particular reaction buffer can betested for its ability to support various reactions by testing thereactions both individually and in combination. The concentration ofcomponents of the reaction such as salt, or magnesium can also affectthe ability of primers or detection probes to anneal to the targetnucleic acid. These can be adjusted in accordance with guidance wellknown in the art, e.g., Innis et al., supra.

C. Exemplary PCR Process

FIG. 8 is a flow chart of a nucleic acid amplification and measurementprocess 800 using apparatus 100. In this example, apparatus 100 controlsa PCR amplification process and detects the presence of multiple targetsequences in the nucleic acid samples.

At step 802, reaction vessels 616 are prepared. Preparation includesplacing reaction components into the vessels and sealing the vessels toprevent spillage or cross-contamination. The reaction components includebuffer, target nucleic acid, appropriate primers and probes,nucleotides, polymerases, as well as optional additional components. Inone embodiment, four fluorescent probes are included, each adapted todetect a different target sequence, and a particular reaction vessel mayinclude any one or more of the fluorescent probes. Each probeadvantageously responds to light of a different incident wavelength andemits light of a different wavelength.

At step 806, a detection module 234 is mounted on shuttle 232. Asdescribed above, detection module 234 may include any number ofdetection channels (i.e., excitation/detection pairs). In oneembodiment, detection module 234 includes four detection channels. Eachchannel is optimized for a different one of the fluorescent probesincluded in reaction vessels 616.

At step 808, reaction vessels 616 are placed into sample wells 210 ofsample unit 202. At step 810, lid assembly 112 is closed and positionedin base unit 110.

At step 812 each channel of detection module 234 is calibrated.Calibration is performed by operating stepper motors 302, 316 toposition detection module 234 such that at least one of its channels isin optical communication with a calibration location 222. As describedabove, each calibration location provides a known fluorescent response.Accordingly, calibration measurements can be used to correct subsequentsample measurements for variations or fluctuations in detector response.Numerous calibration techniques are known in the art. Where detectionmodule 234 has multiple channels, each channel may be independentlycalibrated.

At step 814, a PCR cycle is performed. In general, step 814 involvesoperation of base unit 110 to regulate the temperature of sample unit202, thereby holding the reaction vessels at desired temperatures fordesired lengths of time to complete a two-step or three-step PCR cycle.Base unit 110 may be controlled via user interface 116 or by an externalcomputer.

At step 816, fluorometer assembly 206 scans and interrogates thereaction vessels 616. The operation of fluorometer assembly 206 isadvantageously controlled by an external computer and synchronized withthe operation of base unit 110, so that measurements are identifiable ascorresponding to particular times in the PCR process.

More specifically, at step 816 a, stepper motors 302, 316 or othermotion devices are operated to position detection module 234 such thateach of the four detector channels is in optical communication with adifferent one of sample wells 210 via respective optical windows 502,504, 506, 508. At step 816 b, the LED or other light source for eachchannel is activated (flashed on for a brief period) to stimulatefluorescence. In one embodiment, the LEDs of different channels areoperated in parallel; in an alternative embodiment, they are operatedsequentially so as to avoid reflected LED light from one channel causingfalse signals in the photodetector of another channel.

At step 816 c, resulting fluorescence is detected by the correspondingphotodiode or other detector of the channel, which is read out to theexternal computer. The detectors may be read out in various ways. Forinstance, a peak signal may be detected, the signal may be integratedover a time interval, or the decay of the fluorescent signal after theLED has been deactivated may be measured.

Steps 816 a-c are advantageously repeated, with the position of thedetection module being changed each time so that each channel ofdetection module 234 eventually interrogates each of the sample wells210. In one embodiment, scanning and interrogating four channels foreach of 96 sample wells takes about 15 seconds. The external computeradvantageously executes a program (e.g., the Opticon Monitor programsold by MJ Research, Inc.) that enables a user to view measurement dataas they are collected, in graphical and/or tabular form. Such programsare well known in the art. An example includes the Opticon Monitor™program sold by MJ Research, Inc.

Steps 814 and 816 may be repeated for any number of reaction cycles.Persons of ordinary skill in the art will recognize that real-timefluorescence measurements from process 800 may be used to detect andquantify the presence of each target sequence. Such measurements mayalso be used for purposes such as determining reaction rates andadjusting reaction parameters for improved efficiency, as well asdetermining when additional reaction cycles are no longer needed in aparticular experiment (e.g., when a sufficient quantity of a targetsequence has been produced).

It will be appreciated that process 800 is illustrative and thatvariations and modifications are possible. Steps described as sequentialmay be executed in parallel, order of steps may be varied, and steps maybe modified or combined. For example, fluorescence measurements may beperformed at any point during a PCR cycle, performed multiple timesduring each PCR cycle (including substantially continuous scanning ofthe sample wells), or not performed until after some number of PCRcycles. Any number of distinguishable fluorescent probes may be used ina single reaction vessel, and the detection module may be adapted toinclude at least as many channels as the number of probes in use. Insome embodiments, the detection module includes multiple channelsoptimized for the same probe. This may reduce the scanning time sinceonly one of these channels needs to be used to interrogate a particularsample well.

In addition, as mentioned above, in one alternative embodiment, thevarious channels of detection module 234 are arranged such that when oneof its channels is in optical communication with a sample well 210,other channels are not. This arrangement allows for a “flyover” mode ofoperation, in which detection module 234 is substantially continuouslyin motion during a scanning pass over the wells. Cross-talk between thechannels is reduced because only one sample well at a time receives anyexcitation light.

CONCLUSION

While the invention has been described with respect to specificembodiments, one skilled in the art will recognize that numerousmodifications are possible. For instance, the fluorescence detectionassembly described herein may be adapted for use with a wide variety ofthermal cycler systems and may interrogate sample wells from anydirection (e.g., above or below) in accordance with the design of aparticular instrument. In addition, the system may be adapted to detecta wide range of molecules of biological interest that are identifiableby a fluorescent label or probe; it is not limited to nucleic acids orto any particular amplification process.

Thus, although the invention has been described with respect to specificembodiments, it will be appreciated that the invention is intended tocover all modifications and equivalents within the scope of thefollowing claims.

What is claimed is:
 1. A thermal cycler apparatus comprising: a housing;a sample unit within the housing, the sample unit having a plurality ofsample wells therein; an electronic temperature control subsystemoperable to regulate a temperature of the sample unit such that thesample wells are held at a succession of specific temperatures forspecific lengths of time to complete a reaction cycle; a heater disposedinside the housing and positioned adjacent to the sample unit, theheater having a plurality of openings therein to permit opticalcommunication with each of the plurality of sample wells; and afluorometer assembly disposed inside the housing on a side of the heateropposite the sample wells, the fluorometer assembly including a supportstructure and a movable subassembly movably mounted on the supportstructure, the movable subassembly including a detection module thatincludes: a housing having an opening oriented toward the plurality ofwells; a light emitting diode (LED) light source disposed within thehousing; and an emission light detector disposed within the housing,wherein, during operation of the thermal cycler apparatus, the detectionmodule is movable to be positioned in optical communication withdifferent sample wells of the plurality of sample wells through theplurality of openings in the heater.
 2. The thermal cycler apparatus ofclaim 1 wherein an excitation optical path from the LED light source tothe opening has a fixed length and a detection optical path from theopening to the emission light detector has a fixed length.
 3. Thethermal cycler apparatus of claim 1 further comprising: a calibrationelement disposed such that the detection module is movable so as to bepositioned in optical communication with the calibration element,wherein the calibration element provides a known fluorescence response.4. The thermal cycler apparatus of claim 3 wherein the calibrationelement is located between two or more of the plurality of wells.
 5. Thethermal cycler apparatus of claim 3 wherein the calibration element islocated in an area peripheral to the plurality of wells.
 6. The thermalcycler apparatus of claim 1 further comprising an electronic fluorometrycontrol subsystem operable to move the detection module to interrogateeach of the sample wells at a particular time during the reaction cycle.7. The thermal cycler of claim 6 wherein the electronic fluorometrycontrol system is further operable to coordinate movement of thedetection module and operation of the LED light source and the emissionlight detector such that emission light is measured while the detectionmodule is in motion.
 8. The thermal cycler apparatus of claim 1 whereinoperation of the LED light source and the emission light detector iscontrolled by an external computer.
 9. The thermal cycler apparatus ofclaim 1 wherein the reaction cycle is a polymerase chain reaction (PCR)cycle.
 10. The thermal cycler apparatus of claim 1 wherein the movablesubassembly further includes: a shuttle movably mounted on the supportstructure, the movable detection module being mounted on the shuttle.11. The thermal cycler apparatus of claim 10 wherein the detectionmodule is detachably mounted on the shuttle.
 12. The thermal cyclerapparatus of claim 10 wherein the fluorometer assembly further includesat least two stepper motors mounted on the support structure, thestepper motors being operable to move the shuttle in at least twodimensions.
 13. The thermal cycler apparatus of claim 1 wherein thedetection module includes at least two emission light detectors.
 14. Thethermal cycler apparatus of claim 1 wherein the detection moduleincludes at least two LED light sources.
 15. The thermal cyclerapparatus of claim 1 wherein the detection module further includes aplurality of optical components defining an excitation optical path forlight of an excitation wavelength from the LED light source to theopening of the housing and a detection optical path for light of adetection wavelength from the opening of the housing to the emissionlight detector.
 16. The thermal cycler apparatus of claim 1 wherein thedetection module includes a plurality of excitation/detection pairs,each excitation/detection pair being optically isolated from each otherexcitation/detection pair, and wherein each of the excitation/detectionpairs includes: an LED light source; and an emission light detector.